Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development.biomarker | blood vessels | CNS

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

Background: Exosome vesicles can transfer molecular information previously shown to stimulate tumor development; however, the mechanism of exosome uptake is unknown.Results: Mammalian cells internalize exosomes through lipid raft-mediated endocytosis negatively regulated by caveolin-1.Conclusion: Our findings provide novel insights into cellular uptake of exosomes.Significance: Our data provide potential strategies for how the exosome uptake pathway may be targeted.

Increased vascular permeability is a key feature of inflammatory conditions. In severe infections, leakage of plasma from the vasculature induces a life-threatening hypotension. Streptococcus pyogenes, a major human bacterial pathogen, causes a toxic shock syndrome (STSS) characterized by excessive plasma leakage and multi-organ failure. Here we find that M protein, released from the streptococcal surface, forms complexes with fibrinogen, which by binding to beta2 integrins of neutrophils, activate these cells. As a result, neutrophils release heparin binding protein, an inflammatory mediator inducing vascular leakage. In mice, injection of M protein or subcutaneous infection with S. pyogenes causes severe pulmonary damage characterized by leakage of plasma and blood cells. These lesions were prevented by treatment with a beta2 integrin antagonist. In addition, M protein/fibrinogen complexes were identified in tissue biopsies from a patient with necrotizing fasciitis and STSS, further underlining the pathogenic significance of such complexes in severe streptococcal infections.

Cartilage and tendon extracellular matrices are composed of collagens, proteoglycans, and a number of noncollagenous proteins. Cartilage oligomeric matrix protein (COMP) is a prominent such protein, structurally related to the thrombospondins. We found that native COMP binds to collagen I/II and procollagen I/II and that the interaction is dependent on the divalent cations Zn 2؉ or Ni 2؉ , whereas Ca 2؉ , Mg 2؉ , and Mn 2؉ did not promote binding. Using a solid phase assay, Scatchard analysis identified one class of binding site with a dissociation constant (K d ) close to 1.5 nM in the presence of Zn 2؉ . The results were confirmed by studies using surface plasmon resonance. Furthermore, metal chelate chromatography demonstrated that COMP bound Zn 2؉ and Ni 2؉ . Electron microscopy showed that the interaction occurred at four defined sites on the 300-nm collagen and procollagen molecules. Two were located close to each end, and two at 126 and 206 nm, respectively, from the C-terminal. COMP interacted via its C-terminal globular domain and significantly only in the presence of Zn 2؉ .The major structural constituents of the ECM 1 in cartilage are proteoglycans and collagens. One of the more prominent noncollagenous proteins is COMP. This protein was initially found in articular, nasal, and tracheal cartilage (1), but has later been isolated from tendon (2, 3), where also the corresponding mRNA was demonstrated (3). In the growth plate, COMP is primarily observed in the proliferative region, where it is prominent pericellularly (4, 5), indicating a role in cell growth and matrix development. In more developed articular cartilage, COMP is a major noncollagenous matrix component, primarily located interterritorialy, especially in the more superficial part of the tissue. 2 This high expression of COMP in mature articular cartilage may be induced by the high mechanical load on the tissue. In support, a non-weight-bearing equine tendon shows considerably lower COMP levels than the contralateral weight-bearing one (3).COMP was first isolated from bovine articular cartilage by extraction under denaturing conditions with 4 M guanidine HCl (1). Native COMP has been isolated from Swarm rat chondrosarcoma (6), bovine cartilage (7), and human articular cartilage (8) under mild nondenaturing conditions by extraction with 10 mM EDTA, indicating that the interaction of COMP with components of the ECM depends on divalent cations. Structurally, COMP is related to the thrombospondins (9), having the same molecular domain arrangement of a series of four type 2 (epidermal growth factor) repeat domains followed by seven type 3 domains (calcium binding). COMP, however, is a pentameric glycoprotein consisting of identical 86650 Ϯ 163-Da subunits, each substituted with two N-linked oligosaccarides (10). The five monomers are joined by interactions between their Nterminal portions that form a cylindrical structure (6). The interactions involve the formation of a five-stranded coiled coil (11) from an ␣-helical domain at the N terminus an...